Revisiting two-stage models of chemically-induced cirrhosis-associated hepatocarcinogenesis in rodents: methodological and histopathological approaches

Repeated exposure to hepatotoxins such as carbon tetrachloride (CCl 4 ), thioacetamide (TAA), or diethylnitrosamine (DEN) has been frequently used to induce fibrosis/cirrhosis in rodents. In this narrative review, we compared two-stage medium-term protocols using different CCl 4 or TAA regimens in DEN-initiated rats and mice. In addition, we revisited our previous two-stage DEN/CCl 4 or DEN/TAA rat models to compare fibrosis grade, the mean number, and the percentage of liver area occupied by hepatocellular lesions positive for placental glutathione S-transferase (GST-P). Thus, we evaluated two groups of male Wistar rats that received DEN (a single dose), as an initiator agent for liver carcinogenesis, followed by CCl 4 or TAA regimens (multiple doses) until week 25. Both male Wistar groups presented lower body weight gain and higher liver weight, serum alanine aminotransferase (ALT) levels, hepatocyte proliferation, and a frank development of fibrosis/cirrhosis, GST-P-positive preneoplastic lesions, and liver tumors. However, DEN/TAA protocol in male Wistar rats was more effective as a fibrosis/cirrhosis-associated hepatocarcinogenesis model since TAA effectively accelerates liver disease progression and carcinogenesis. In addition, these chemical regimens have been prospectively studied as a suitable and effective protocol that resembles the corresponding human disease with various molecular and morphological outcomes. In conclusion, two-stage DEN/CCl 4 or DEN/TAA rodent models are effective in mimicking the progression of fibrosis-associated hepatocarcinogenesis and are suitable for prophylactic or therapeutic investigations.